The Efficacy of Targeted Monoclonal IgA Antibodies Against Pancreatic Ductal Adenocarcinoma

Abstract

The efficacy of immunotherapy in pancreatic ductal adenocarcinoma (PDAC) remains limited. The tumor microenvironment (TME), characterized by the accumulation of suppressive myeloid cells including neutrophils, attributes to immunotherapy resistance in PDAC. IgA monoclonal antibodies (mAbs) can activate neutrophils to kill tumor cells; this can be further enhanced by blocking the myeloid immune checkpoint CD47. In this study, we investigated the potential of this therapeutic strategy for PDAC. We determined the expression of tumor-associated antigens (TAAs) on PDAC cell lines and fresh patient samples, and the results showed that the TAAs epithelial cell adhesion molecule (EpCAM), trophoblast cell surface antigen 2 (TROP2) and mucin-1 (MUC1), as well as CD47 were consistently expressed on PDAC. In line with this, we showed that IgA mAbs against EpCAM can activate neutrophils to lyse various PDAC cell lines and tumor cells, which can be augmented by addition of CD47 blockade. In addition, we observed that neutrophils were present in patient tumors and expressed the receptor for IgA. In conclusion, our results indicate that a combination of IgA mAb with CD47 blockade is a promising preclinical treatment strategy for PDAC, which merits further investigation.

Keywords: IgA; PDAC; PMNs; mAbs; monoclonal antibodies; neutrophils; pancreatic cancer; pancreatic ductal adenocarcinoma; polymorphonuclear cells.

Figure 1

Processing of patient tumors.

Figure 2

Tumor-associated antigens (TAAs) expressed on pancreatic ductal adenocarcinoma (PDAC) cell lines and efficacy of IgA monoclonal antibodies (mAb) targeting these TAAs in antibody-dependent cellular cytotoxicity (ADCC) assay. (A) Mean fluorescent intensity (MFI) of TAA and CD47 expression as measured with flow cytometry. Indirect staining was performed with 10 µg/mL target IgA antibody and for CD47 a PE-conjugated antibody was used. 3 independent experiments are shown in 1 representative graph. (B) MFI of mucin-1 (MUC1) expression as measured with flow cytometry. Indirect antibody staining was performed with 10 µg/mL 214D4 antibody (Ab) or 10 µg/mL 139H2 Ab. 3 independent experiments are shown in 1 representative graph. (C) Antigen density of EGFR, EpCAM, TROP2 and CD47 was determined with QIFIKIT analysis using 10 µg/mL target antibody. Mean ± standard error of the mean (SEM) of specific antibody binding capacity (SABC) is shown of a minimum of n = 3 independent experiments. (D–H) Tumor cells were labeled with chromium-51 (51Cr) for chromium release assays and subsequent chromium release was measured after 4 h of incubation with antibodies and whole leukocytes (WL) or polymorphonuclear leukocytes (PMN). For CD47 block, target cells were incubated with IgG1 PGLALA SIRPα fusion protein prior to 51Cr release ADCC assay. (D) Panc 10.05 ADCC with WL comparing IgA with IgG mAb against EpCAM and (E) against TROP2. (F) PMN ADCC of Panc 10.05, (G) Capan-2 and (H) CFPAC-1 cell lines with IgA mAb targeting EpCAM (heING1), EGFR (cetuximab) and TROP2 (sacituzumab). The Mean ± SEM of specific lysis is shown of at least n = 3 independent experiments. Independent experiments are performed in technical triplicate. Two-way ANOVA followed by Tukey’s post-hoc test was performed. ** p < 0.01, **** p < 0.0001.

Figure 3

Tumor-associated antigens (TAAs) and CD47 expression on tumors from patients with pancreatic ductal adenocarcinoma (PDAC). Digested tumor samples were measured with flow cytometry after RBC lysis. Dead cells (To-Pro-3) and leukocytes (CD45+) were excluded. Exclusion of all fibroblasts was not possible and we assumed all tumor cells expressed the epithelial marker EpCAM. (A) Bar graphs showing the percentage of TAA positive cells relative to EpCAM. (B) Bar graphs depicting the mean fluorescent intensity (MFI) of the TAA positive cell populations. (C) Heatmap depicting the relative expression (based on MFI) of different TAAs and CD47 on PDAC patient tumor cells, red is high expression, orange is low expression and blue is no expression. The TAAs depicted in white were not determined. The targets are represented as measured, however low viability and cell numbers can influence the results and cause false negatives.

Figure 4

Efficacy of EpCAM-IgA against patient tumor cells. Polymorhonuclear leukocyte (PMN) antibody-dependent cellular cytotoxicity (ADCC) assay with IgA against EpCAM and pancreatic ductal adenocarcinoma (PDAC) tumor cells isolated via negative MACS selection (CD45, CD31, PDGFRα- and PDGFRβ negative) after red blood cell lysis. Lysis of patient tumor cells was corrected to percentage of isolated tumor cells since not all fibroblast could be excluded from the sample. EpCAM expression in (A) tumor sample #10 and (C) #14 after tumor cell isolation. ADCC of Panc 10.05 cell line and tumor cells from (B) patient sample #10 and (D) #14. The mean ± SEM of specific lysis of a technical triplicate is shown. Two-way ANOVA followed by Tukey’s post-hoc test was performed. **** p < 0.0001.

Figure 5

Characterization of cell types in patient tumor samples Patient tumors were digested to a single cell suspension and further analyzed with flow cytometry. (A) Patient tumor samples contain red blood cells (RBCs, CD235a), leukocytes (CD45) and tumor or epithelial cells (EpCAM). For further analysis red blood cell lysis was performed. Dead cells were excluded with 7-AAD as live-death marker. (B) Leukocyte composition evaluation in tumor sample shows the presence of granulocytes (PMN), monocytes, T, B, and NK cells. (C) Staining for the Fc alpha receptor (FcαR) and (D) LOX-1 was performed on PMN.